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Magnesium chloride

氯化镁

Company: Sigma-Aldrich
Catalog#: M8266
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Detecting the Interaction of Double-stranded RNA Binding Protein, Viral Protein and Primary miRNA Transcript by Co-immunoprecipitation in planta
Author:
Date:
2018-05-05
[Abstract]  MicroRNAs (miRNAs) play important roles in plant growth, development, and response to infection by microbes. Double-stranded RNA binding protein 1 (DRB1) facilitates the processing of primary miRNA transcripts into mature miRNAs. Recently, we found that NS3 protein encoded by rice stripe virus (RSV) associates with DRB1 and promotes miRNA biogenesis during RSV infection (Zheng et al., 2017). RNA co-immunoprecipitation (RIP) method was applied to identity association patterns among DRB1, NS3, and miRNA transcript. [摘要]  微小RNA(miRNA)在植物生长,发育和微生物感染反应中发挥重要作用。 双链RNA结合蛋白1(DRB1)有助于将初级miRNA转录物加工成成熟的miRNA。 最近,我们发现水稻条纹病毒(RSV)编码的NS3蛋白与DRB1相关并促进RSV感染期间的miRNA生物合成(Zheng等人,2017)。 RNA共免疫沉淀(RIP)方法被用于鉴定DRB1,NS3和miRNA转录物之间的关联模式。

【背景】在双链RNA(dsRNA)结合蛋白HYPONASTIC LEAVES1(DRB1 / HYL1)的帮助下,通过RNA酶III酶DICER-LIKE1(DCL1)从其初级转录物(pri-miRNA) 锌指蛋白SERRATE(SE)。 水稻条纹病毒(RSV)感染广泛地干扰miRNA积累。 我们发现RSV编码的非结构蛋白3(NS3)通过与水稻中的DRB1相互作用下调pri-miRNAs来促进miRNA积累(Zheng等人,2017)。 为了揭示NS3如何增强pri-miRNA的加工,我们使用免疫共沉淀(Co-IP)来说明NS3,DRB1和pri-miRNA体内的关系。 该协议有助于了解两种蛋白质和一种RNA转录本之间的关联模式。

Metal-tagging Transmission Electron Microscopy for Localisation of Tombusvirus Replication Compartments in Yeast
Author:
Date:
2018-04-20
[Abstract]  Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and host factors and create an environment that facilitates the formation of VRCs within replication organelles. Therefore, efficient virus replication depends on the assembly of specialized membranes where viral macromolecular complexes are turned on and hold a variety of functions. Detailed characterization of viral replication platforms in cells requires sophisticated imaging approaches. Here we present a protocol to visualize the three-dimensional organization of the tombusvirus replicase complex in yeast with MEtal-Tagging Transmission Electron ... [摘要]  正链(+)RNA病毒是人,动物和植物中的细胞内病原体。构建病毒复制酶复合物(VRC)病毒操纵脂质流动和重组亚细胞膜。重新设计的膜集中了病毒和宿主因子,并创造了促进复制细胞器内VRC形成的环境。因此,有效的病毒复制取决于病毒大分子复合物开启并具有各种功能的特殊膜的组装。细胞中病毒复制平台的详细特征需要复杂的成像方法。在这里我们提出一个协议,用肉眼标记透射电子显微镜(METTEM)可视化酵母中的tombusvirus复制酶复合物的三维组织。该协议使我们能够用METTEM和电子断层扫描成像三维病毒复制酶分子的细胞内分布。我们的研究显示病毒复制酶分子如何在特化细胞膜内构建复制复合物。

【背景】正链RNA病毒的复制取决于细胞膜的重塑。细胞内膜作为VRC装配的结构支架,提供调节病毒复制酶活性和保护病毒RNA免受宿主抗病毒防御的必需脂质和辅因子(Miller和Krijnse-Locker,2008; den Boon <等,2010; nagy和pogany,2011;="" nagy,2016)。电子显微镜观察到具有活性vrc的复制细胞器的结构。="" vrc以单个膜囊或'小球',管状球形立方体膜,双膜囊泡(dmv)或平面寡聚体阵列装配(de="" castro等人,2013)。通常在rna病毒感染的细胞中观察到小球。它们通过在各种细胞器中内陷而形成,并具有对胞质溶胶的狭窄开口(den="" boon="" et=""> ...

Purification of RNA Mango Tagged Native RNA-protein Complexes from Cellular Extracts Using TO1-Desthiobiotin Fluorophore Ligand
Author:
Date:
2018-04-05
[Abstract]  A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose beads. The beads are washed to remove non-specific complexes and then the RNA Mango containing complexes are eluted by the addition of free biotin to the beads. Since the eluted complexes are native and fluorescent, a second purification step such as size exclusion chromatography can easily be added and the purified complexes tracked by monitoring fluorescence. The high purity native complexes resulting from this two-step purification strategy can ... [摘要]  描述了使用RNA Mango进行RNA-蛋白质复合物的RNA纯化的天然纯化策略。 RNA芒果适体首先被基因工程改造成感兴趣的RNA。 然后将从清除的细胞天然提取物获得的含有RNA复合物的复合物固定在TO1-Desthiobiotin饱和的链霉亲和素琼脂糖珠上。 洗涤珠粒以去除非特异性复合物,然后通过向珠粒中加入游离生物素来洗脱含RNA芒果的复合物。 由于洗脱的复合物是天然的和荧光的,所以可以容易地添加第二纯化步骤如尺寸排阻色谱,并且通过监测荧光追踪纯化的复合物。 通过这种两步纯化策略产生的高纯度天然复合物可以用于进一步的生物化学表征。

【背景】目前的RNA标签受限于诸如差K ,大尺寸,潜在的生物学干扰或缺乏固有荧光的限制(Panchapakesan等人, 2015年)。 RNA芒果很小,可以简单地整合到茎环结构中,特别是GNRA tetraloops中,它具有生物耐受性,并且最重要的是对其噻唑橙基(TO1)配体TO1-Desthiobiotin(TO1-Dtb)具有高亲和力。 ...

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